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Histochemical stains-2

 MR G.P. TIWARI
 Tata Memorial Hospital

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Outline of the lecture
 Technical aspects of histochemical stains
and its application
 Stain principle
 Control source
 Procedures and preparation
 Its results
 basic troubleshooting

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AFB
The Ziehl-Neelsen stain, also
known as the acid-fast stain, was
first described by two German
doctors; Franz Ziehl a bacteriologist
and Friedrich Neelsen a
pathologist. IN 1882

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Application
IT IS USED TO DEMONSTRATION
OF ACID FAST BACILLI BELONGS
TO GENUS MYCOBACTERIUM
TUBERCULOSIS AND M. LAPRAE.
CONTROL: AFB CONTAINING
TISSUE
Principle
Mycobacterium( mycolic acid and lipids)

These are ß-hydroxy carboxylic acids

it has a chain lengths of 90 carbon atom

The acid-fast ness depend on


ability of mycobacterium to retain dye

acid solution
Preparation of reagents
1) Zeil Neelson carbol fuchsin solution
Basic fuchsin--- 1 gm
Absolute alcohol-10ml
5% phenol solution-100ml (5 grams phenol dissolved in
100ml of distilled water Mix well. Filter into brown
color bottle.
2)1% acid alcohol
3)1% Methylene blue

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PROCEDURE

1) Bring section to water.


2) Filter ZNCF solution over the sections for 30
minutes.
3) Wash with water for 10 minutes.
4) Differentiate with1% acid alcohol till
decolorized.
5) Wash well under running water
6) Counterstain with 1% Methylene blue-1 dip
7) Wash, dehydrate, clear and mount in DPX.

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Results

Acid fast bacilli-bright red


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Background-light blue 8
Improper decolorization

It will lead to false positive


results to overcome this problem
proper decorization must carried
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FITE AND FARACO
MODIFICATION

IT WAS IN YEAR 1940 FITE AND


FARACO HAVE MODIFIED AFB
TECHNIQUE FOR DELICATE
MYCOBACTERIUM LEPRAY BACILLI

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MODIFICATION
    MYCOBACTERIUM LAPREY BACILL IS LESS
ACID FAST THEN AFB

DEPARAFFINIZATION WITH MINERAL


OIL( 2PART OIL 1PART XYLENE)

5% H2SO4 SOLUTION (DECOLORIZATION)

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Remark
We must filter the ZNCF stain before staining
with watman filter paper
Proper decorization must be carried out
After this thorough washing is required to
remove acid.
We must filter methylene blue with watman
filter paper during counterstaine
Counterstaining should be light otherwise it
will mask the bacilli.
This procedure can be performed either using
heat or without using heat

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GMS
It was first described
by Gomori in1937
later on it was
modified by Grocotts
in 1955
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APPLICATION

This technique is
mainly used to
identify fungi in the
sections
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Principle
The mucopolysaccharide component of
fungal cell wall is oxidized to release
aldehyde groups. The aldehyde groups
react with silver nitrate and silver stain
precipitates along the fungal walls.

CONTROL: Any tissue containing


fungus.

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SILVER INPREGNATION

 The oxidation of aldehyde may be carried out by


5% chromic acid or by 0.5% periodic acid
 The aldehyde group of reticulum is reduced the
colorless silver complex , to dark brown ,form
lower oxides of silver
 This lower silver oxide is reduced to black
metallic silver by 10% formalin
 The unreduced silver is removed by sodium
thiosulphate
 Toning gives pale grey back ground which is
better for photography and counter stain
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Principle

Fungal cell wall (mucopolysaccharide)


Oxidation
Chromic acid
Aldehyde groups
Reduction
Silver stain
Black precipitate along fungal cell wall

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Preparation of reagents

 Stock solution – silver methanamine soln


 5ml 5% silver nitrate + 100ml 3% methenamine
(Hexamethelene tetramine)
 Store in Refrigerator

 Working solution
 25ml stock solution + 25ml distilled water + 3ml 5%
borax solution.

 Always prepare fresh working solution

 
 
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Gomori/ Grocotts Modification
Oxidize with chromic acid at room temperature for 1 hour or by
periodic acid for 10 min

2% sodium metabisulphite for 2 min( to remove chromic acid)


(Reduction)
silver methanamine sol. ( 24 hrs at R.T, / 1hrs at 60’c)

Gold Chloride 2 min( toning)

Sodium thiosulphate 2 min( remove excess silver stain)

counter stain Light Green 30 sec


Results-

 Fungus-black
12/08/21  Background-light green 20
Remarks

If chromic acid turns brown, prepare fresh


solution

It is mandatory to use fresh working


solution each time.

Avoid higher temperature as this causes


background staining.

After every step, rinse the slide in distilled


water
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Troubleshooting

Light staining if incomplete reduction


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Troubleshooting

Dark staining if over-reduction


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Prussian Blue
It was first described by perls in 1867
it demonstrated hemosiderin
pigments in the tissue. it is a golden
brown pigment it arise when
hemoglobin broken down in the tissue
Control :A known positive control
tissue of hemorrhagic lesion

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application
1.Bone marrow – Iron deficiency
anemia, Myelodysplastic syndrome,
Refractory
anaemias.
2. Hemosiderosis-due to excess
therapeutic iron or blood transfusion,
demonstrated
in spleen, bone marrow, liver.
3. Hemachromatosis
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Principle
Ferric iron
of the hemoglobin

+
2% ferrocyanide in2% hcl

Ferric ferrocyanide

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Procedure

2%Potassium 30
2% conc. HCL
ferocyanide min

Wash thoroughly with distilled


water

1% Eosin

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Prussian Blue
 Demonstration of hemosiderin pigment.

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Alizarin Red S Method

Demonstrations of calcium pigment

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Alizarin Red S Method
It was first introduce by
mc.gee –Russell in1958
it is used to
demonstrated calcium
salts in the tissue

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Principle

Calcium forms an Alizarin red S- calcium


complex in a chelating process. The orange
red precipitate formed is birefringent
Control:
Known calcium containing tissue
section .

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application

This method is useful in


identification and detection of
small amounts of calcium like
in hypercalcinosis in kidney, in
hyperthyroidism necrosis, in
some myeloma and schwamoma
body
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Principle
Calcium

Alizarine red S

Alizerine red S
calcium complex (orange red is formed)

Birefringent in polarized
light
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Procedure

Air dry section

Alizarine red S
(5min)PH-4.2

Dry section with acetone

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GIEMSA

It was first described


by Gustav Giemsa,
an early malariologist
in 1902
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Giemsa stain
 Introduction-
 Differentiation of cells of hemopoietic
tissue
 demonstrates some microorganisms like
h-pylori , trypanosoma ,and parasites
malaria, in peripheral blood smears . And
for bone marrow and chromosomes
structure

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Principle

Giemsa-
1.Methylene blue-stains
acidic cell components
2.Eosin-stains basic cell
components
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Preparation of reagents
1 Gms Giemsa powder
+54 ml Glycerin
preheated 60 0 C.+ add
84ml Methanol. Mix well
and filter the stain
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Preparation of reagents cont
 Buffer solution
 Potassium dihydrogen phosphate—2.72 gms
 Distilled water— 100 ml
 Sodium hydroxide – 0.8 gm
 Distilled water – 100ml
 50 ml of Potassium dihydrogen phosphate is
mixed with 23.6 ml of NaOH. The pH of the
solution is adjusted to 6.8.

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Procedure

 Giemsa stain must be diluted with buffer


solution in1:9 ratio (Working) 20-30
minuets.
 Differentiate with 0.2 % acetic acid quickly
and dip in tap water briefly

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Procedure for bone marrow
imprints &smears
 Smears are fixed in methanol for 30 min.
 Stain in working Giemsa solution 30
minutes.
 Wash in water for 5 minutes.
 Air dry smears.
 Clear in Xylene , mount in DPX.

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Result – Bone Marrow Imprint

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RESULTS

12/08/21 H.Pylori organisms in gastric glands 43


Results

 H. Pylori – pinkish blue


 Mast cells—Magenta pink
 Tissue elements—Shades of blue to pink

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Gram Staining
The method is named after its
inventor, the Danish scientist
Hans Christian Gram who
developed the technique in
1884

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application
The purpose is to
demonstrate Gram negative
& Gram Positive organisms
in the tissue.

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Principle OF Grams stain
Gram +ve Insoluble lake
barrier

Gram +ve

Crystal violet Legols iodine acetone Basic fuschin

Lipo poly
saccharide

Gram -ve Gram -ve

pepidoglycan

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BROWN AND BRENN
Crystal violet (1 min)

Dry section with acetone


Lugol’s iodine
(1min)
Picric acid- acetone
Acetone (until no
colour)

Acetone (2dip)

Basic fuschin (3min)

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BROWN AND BRENN

ORAL FLORA
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Melanin

It was first described


by Massons in 1912
later it is modified by
Fontana in1914

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application
For the detection of Melanin
pigments and Argentaffin
granules can be demonstrated by
this method. Melanin is an
intracellular pigment it is usually
located in skin retina substantia
nigra of brain and in hair.
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Principle

 Melanin

silver Solution at 60’C

reduction

Black coloured metallic silver

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Procedure
    Silver solution 600C for 1 hour.

0.2% Gold chloride

2% Sodium thiosulphate

1% Eosin

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Prepration of silver nitrate
 5ml 10% Silver nitrate
+
 45 ml distilled water (1 hr at 600c )

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RESULTS

Melanin Pigment In Basal Layer Of


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Skin 55
Melanin bleach method
 Procedure-

0.5 Potassium permanganate


2% oxallic acid
Routine H&E staining

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Precautions
 Check under microscope, if required
repeat
 Bleaching step
 Preferably use Poly-L- Lysine coated
slides.

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MELANIN BLEACH

PIGMENTED MELANOMA
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Chloroacetate esterase
Pararosaniline method
 Introduction :
Chloroacetate esterase is the only enzyme
which can be demonstrated in paraffin
section.

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Principle
Leukocyte esterase

hydrolyses

Derivative of Naphthalene
+
Diazonium salt

Brightly coloured ppt at the site of enzyme activity

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Preparation of reagents

 1) substrate solution :
Naphthol As. D. Chloroacetate- 10 ml
 N-N dimethyl formamide –1 ml:
 Pararosanilin– 0.4gms, distilled water – 0.4ml, conc.
HCl – 1.6ml.
 sodium nitrite – 0.4gms, distilled water – 10ml.
 Concentrated HCl – 8.35 ml ,distill water 91.65 ml.
 Sodium barbital powder – 1.03gm, distilled water –
100ml.

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Procedure

     14.6ml of 0.1N HCL and 15.4ml of sodium barbital.


     prepare substrate solution and keep aside. Drop
of Pararosaniline and add drop of sodium nitrite.
Add all solutions adjust pH 6.3
Filer and keep for 3hr
counter stain with hematoxylin.

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Result
 Reactive lymph node -neutrophils

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